The present invention concerns a simplified process for the solubilization and renaturation of denatured proteins, in particular of recombinantly produced denatured proteins.
Sparingly soluble inactive protein aggregates (inclusion bodies) are frequently formed when proteins are produced in prokaryotic cells such as E. coli. In order to convert these proteins into their active form it is necessary to solubilize and to renature these proteins. Such processes are known and are for example described in EP-A 0 361 475, EP-A 0 114 506, EP-A 0 093 619, EP-A 0 253 823, WO 87/02673, EP-A 0 364 926 and EP-A 0 241 022. An important factor in the activation which limits the yield of renatured protein is the competing reaction between conversion of the renatured protein into the correctly folded intermediate and an aggregation of several protein molecules. For this reason the concentration of renatured protein in the renaturation solution is an important parameter for the yield of the renaturation process. Aggregation is favoured by increasing concentrations of renatured protein and the relative yield of renatured protein with the conformation of the native protein decreases (critical concentration).
In a large-scale production of recombinant proteins the amount of protein to be renatured is usually much higher than the critical concentration. Since the proteins often have a low solubility in the activation buffer used, this therefore results in considerable disadvantages such as low yield, long time requirement and large buffer volumes.
A process is known from WO 87/02673 in which the inactive soluble protein is solubilized with denaturing agents and reducing agents, subsequently the reducing agent is separated and then heterologous mixed disulfides between protein and for example glutathione are prepared from the solubilized proteins. Such mixed disulfides are advantageous for the further purification and renaturation since after modification of the thiol groups the protein is protected against air oxidation and it is thus stable in a larger pH range. The change in the net charge also facilitates the purification since it enables non-modified proteins to be separated by means of ion exchange chromatography.
In order to form the mixed disulfides, the solubilized, dialysed and reduced protein that has been purified of reducing agents is incubated with a solution which contains a denaturing agent and a disulfide component for the derivatization (e.g. GSSG, cystine, cystamine). A renaturation is carried out in the usual manner after separation of the disulfide component. Although this process is efficient, it requires many individual process steps especially for separation of the reducing agent before the derivatization.
A process for folding and purifying insulin-like growth factor I is known from WO 93/19084. According to this the inclusion bodies are dissolved under reducing conditions, subsequently an excess of oxidizing agent is added without separating the reducing agent. The renaturation is initiated by subsequent dilution (without dialysis) and new addition of reducing agent (to construct a redox system). WO 91/08762 describes the preparation of biologically active platelet derived growth factor. In this process a solubilization is firstly carried out at pH 3 without a reducing agent and subsequently a purification under denaturing conditions. Only afterwards is an oxidizing agent added to produce a derivative. According to EP-A 0 450 386 an extract of inclusion bodies (denatured dissolved NGF protein) is prepared by adding solubilizing buffer with subsequent centrifugation. The extract is then treated with a reducing agent, incubated and oxidized by addition of an oxidizing agent without previous dialysis. Subsequently it is diluted and further components are added for the denaturation. Thus in this process a solubilization is firstly carried out as a separate process step without adding redox active substances. None of these processes is suitable for a pulse renaturation according to U.S. Pat. No. 4,933,434.
In addition processes are known which already allow a derivatization during the solubilization. The method of sulfitolysis has been known for a long time (e.g. Bailey, J. L., Cole, R. D., 1959, J. Biol. Chem. 234, 1733-1739; Cole, R. D., 1967, In: Meth. Enzymol. 11, 206-208; EP 0 114 507). In this process disulfide bridges in proteins are treated with salts of sulfurous acid to form a mixture of 50% thio-sulfonated (RS-SO-xe2x88x923) and 50% free (RSxe2x88x92) protein-SH groups as the reaction product. The latter free SH groups are in turn converted into disulfides by reoxidation (e.g. with copper ions, iodosobenzoate or preferably with tetrathionate) which can be almost completely converted into the thiosulfonate by repeated cycles of the process. This process is relatively simple and can be carried out under mild boundary conditions (e.g. neutral pH value). As already set forth in J. Biol. Chem. 234, 1733 a disadvantage is that the thiosulfonate that is formed is chemically labile, it is not possible to check the completion of the conversion and above all the tryptophan residues are partially destroyed by the reoxidation agent. A further disadvantage is that it is very difficult to completely separate by-products containing thiosulfonated protein-SH groups and oxidizing agents such as the said iodosobenzoate in the final product and it is extremely laborious to detect this analytically. However, this is absolutely necessary for proteins which are intended for a therapeutic application in order to exclude possible side effects of a therapeutic agent that has been chemically modified in such an unphysiological manner.
WO 95/30686 also describes such a sulfitolysis to renature neurotrophic factors of the NGF/BDNF family. A similar process is described for the renaturation of human proinsulin by R. Wetzel et al., Gene 16 (1981) 63-71 as well as by W. F. Heath et al., J. Biol. Chem. 267 (1992) 419-425.
A similar method which avoids the use of reoxidation conditions that damage side chains is also known (Thannhauser, T. W., Konishi, Y., Scheraga, H. A., 1984, Analyt. Biochem. 138, 181-188; Thannhauser, T. W., Scheraga, H. A., 1985, Biochemistry 24, 7681-7688): In this case instead of a reoxidation, the cysteine obtained when the disulfide bridge is reduced is directly derivatized by reaction with 2-nitro-5-(sulfothio)-benzoate; 2-nitro-5-thiobenzoate is released in this process which can be measured photometrically and thus enables a quantification of the converted SH groups. A disadvantage of this method is that a complex chemical substance is introduced whose complete separation from the final product is very time-consuming and difficult to check. Furthermore the authors (Biochemistry 24, 7681) observed that the thiosulfonate obtained is only stable when thiol groups are completely absent. In addition a side chain modification is also observed in this case (deamination of asparagine).
The object of the present invention is to simplify and to improve these processes and to provide stable, storable proteins whose SH groups are derivatized and which can be renatured in a high yield.
Surprisingly it was found that the process according to the invention allows the solubilization and derivatization to be carried out in a single step without having to previously reduce. It is particularly surprising that the derivatization can also take place under acidic conditions (pH value less than 7.0, preferably pH 3-6.5) preferably for neurotrophins such as NGF and that this is achieved without essentially affecting the kinetics and the completeness of a reaction compared to a reaction with thiol components in the usual pH range of about 7-10. It is usually assumed that such reactions can only proceed in the presence of the free thiolate anion; this only occurs in effective concentrations at pH values above about 7 due to the high pK value of thiolate anions of about 9.
The invention therefore concerns a process for producing mixed disulfides composed of a protein and a disulfide component which is characterized in that the protein in an inactive sparingly soluble form (inclusion bodies) is incubated, dissolved and derivatized with a solution of a denaturing agent in a denaturing concentration and in the presence of a disulfide component (molar ratio protein : disulfide component 1:1 to 1:10000, preferably 1:1000) and subsequently the disulfide component is optionally removed. The disulfide component can then be expediently removed by subsequently carrying out a pulse renaturation as described in the U.S. Pat. No. 4,933,434. The derivatized protein according to the invention is stable and can be stored before further processing. This is particularly advantageous since the derivatized protein can be produced by the process according to the invention independently of the renaturation. Hence the derivatized protein is available as an isolated intermediate product for numerous renaturation and purification processes and/or preparations.
Alternatively the incubation to derivatize the protein is carried out in the presence of a reducing agent (e.g. DTT, DTE, GSH, cysteine, cysteamine, salts of sulfurous acid). This can improve the derivatization yield. In this case it is expedient to select the concentration of the reducing agent so that the effectiveness of the disulfide component is not restricted or only to a slight extent; reducing agent concentrations of up to 20 mole percent, preferably up to 10% of the concentration of the disulfide component have turned out to be favourable.
Additional reagents can be preferably added to protect free SH groups which can partially or completely prevent blocking or destruction of these SH groups by heavy metals, radicals or active oxygen species. This class of protecting reagents for example includes EDTA at a concentration of 0.1 to 100 mmol/l or mannitol at a concentration of 1 to 1000 mmol/l.
Disulfide components are understood as substances from the disulfide class e.g. GSSG, cystamine or cystine. Disulfide components are able to derivatize SH groups in proteins after cleavage of a disulfide bridge. The disulfide component is preferably used at a concentration of at least 1 mmol/l or higher, preferably of 1-1000 mmol/l, particularly preferably of 10-200 mmol/l.
It is expedient to use a denaturing agent that is usually used to solubilize denatured protein under oxidizing conditions as the denaturing agent. It is preferable to use guanidinium hydrochloride or other guanidinium salts such as e.g. sulfate, phosphate or thiocyanate as well as urea or derivatives thereof. It is also possible to use mixtures of these denaturing agents.
The concentration of the denaturing agent depends on the type of the denaturing agent and can be easily determined by a person skilled in the art. The concentration of the denaturing agent is adequate if a complete solubilization of the denatured sparingly soluble protein can be achieved. In the case of guanidine hydrochloride these concentrations are usually 3-8 mol/l, preferably 6-8 mol/l. In the case of urea the concentration is usually 6-10 mol/l.
xe2x80x9cProtein in an inactive sparingly soluble formxe2x80x9d is understood as a protein which is for example formed by recombinant production in prokaryotes. Such proteins are usually formed when eukaryotic proteins are overexpressed in prokaryotes and the protein is not transported in an active form into the periplasm or into the cell supernatant. In this case the recombinantly produced protein remains in the cytoplasm or periplasm in an insoluble and aggregated form. Such aggregates, their isolation and purification are described for example in Marston F. A. O., Biochem. J. 214 (1986) 1-12. The prokaryotic cells are lysed after the fermentation to isolate inclusion bodies.
The cell lysis can be carried out according to the usual methods e.g. by means of ultrasound, high pressure dispersion or lysozyme. It is preferably carried out in a buffer solution that is suitable for adjusting a neutral to weakly acidic pH value as a suspension medium such as e.g. 0.1 mol/l Tris-HCl. After cell lysis the insoluble components (inclusion bodies) are separated in any desired manner, preferably by centrifugation or by filtration after washing with agents which do not interfere with the proteins but dissolve foreign cell proteins as completely as possible e.g. water or phosphate buffer optionally with addition of mild detergents such as Brij(copyright). Subsequently the precipitate (pellet) is subjected to the process according to the invention for solubilization and derivatization.
The process according to the invention is carried out in a neutral to alkaline pH range, preferably between pH 6 and 10, particularly preferably in the pH range between 7 and 8. All common buffers are suitable as buffer solutions; when guanidinium hydrochloride is used as a denaturing agent it is not necessary to add buffers because of its buffering action. Buffers known to a person skilled in the art are preferably used such as e.g Tris or phosphate. Surprisingly the process according to the invention can also be particularly advantageously used for neurotrophins even under acidic conditions (pH 3-6.5).
The process according to the invention is carried out with the addition of a disulfide component. Preferred disulfide components are e.g. GSSG, cystamine and cystine. Since the derivatization reaction is an equilibrium reaction between protein in the thiol form and the disulfide component or between a mixed disulfide composed of protein and disulfide component on the one hand and the free thiol components i.e. remaining protein thiol groups and, on the other hand, thiol components released from the disulfide component by reaction with the protein in the thiol form, the desired derivatization reaction must be forced by a high excess of the disulfide component. The conditions that are necessary for this are very different from protein to protein. It is preferable to use a concentration range of the disulfide component from 10 mmol/l up to the saturation limit (e.g. ca. 200-300 mmol/l in the case of GSSG depending on the pH value of the preparation, ca. 700 mmol/l for cystamine), the concentration range is particularly preferably 50-100% of the saturation concentration of the disulfide component.
It is also preferable to add a reducing agent in the process according to the invention. Reducing agents from the mercaptan group are particularly preferred such as reduced glutathione (GSH) or 2-mercaptoethanol, dithioerythritol (DTE) or dithiothreitol (DTT) at a concentration of 0.01-50 mmol/l, preferably 0.1-10 mmol/l. Reducing agents such as salts of sulfurous acid e.g. sodium sulfite are additionally preferred. Although the addition of one of these reducing agents is not a prerequisite for successfully carrying out the reaction, this addition can, however, lead to an improved yield when the protein is reactivated depending on the treated protein.
The process according to the invention is preferably carried out at room temperature during a period of 0.1-100 hours, preferably 1-24 hours, particularly preferably 2-4 hours. Other conditions such as heating to about 60xc2x0 C. or a process with cooling to about 0xc2x0 C. are, however, also suitable. In order to prevent oxidation of the reducing agent by atmospheric oxygen and to protect free SH groups, it is expedient to add EDTA preferably in an amount of 1-100 mmol/l, particularly preferably by ca. 10 mmol/l. In order to suppress radical side reactions which can for example occur in solutions containing thiol especially at relatively high pH values it is also expedient to add radical interceptors (quenchers) such as e.g mannitol at a concentration of 1 to 1000 mmol/l, preferably at a concentration of 20 to 200 mmol/l, particularly preferably at a concentration of 50 mmol/l during the renaturation and/or processing of the proteins.
After solubilization/derivatization it is preferable to dialyse against a solution which contains a denaturing agent in a denaturing concentration in order to remove the disulfide component and optionally added reducing agent. The dialysis solution advantageously contains the denaturing agent at the same concentration as in the denaturation/derivatization solution. It is also preferable to dialyse against other denaturing agents at the same molar concentration, e.g. against ca. 1 mmol/l HCl or dilute acetic acid. Furthermore it has also turned out to be expedient to not completely separate the disulfide component; as already explained the derivatization reaction is an equilibrium reaction between free thiol components and (optionally mixed) disulfide components. If all protein thiol groups have not been completely derivatized, there is a risk after separation of the disulfide component that the remaining free thiol components will have a reducing effect on the mixed disulfides that are present and hence that the derivatization yield will subsequently decrease during storage of the derivatized protein. The degree of derivatization of the treated protein must therefore be as high and stable as possible with regard to the intended aim of the derivatization to protect the protein thiol groups against oxidation and against similar destructive side reactions. This can either be achieved by prematurely terminating the dialysis before the concentration of the disulfide component has fallen below a suitable concentration or by dialysing in the dialysis against a dialysis buffer which contains the disulfide component at the required concentration. The concentration required to maintain the degree of derivatization during storage of the derivatized protein depends on the respective treated protein and in particular on the cysteine content of the treated protein and can be in a concentration range of 0-100 mmol/l. With regard to the further use of the derivatized protein for the reactivation reaction, care must be taken that the introduction of the disulfide component in the reactivation process has no or only a negligible effect on the conditions used in this case for the desired oxidative linkage of intermolecular or intramolecular disulfide bridges. For this reason a residual concentration of disulfide component in the derivatized protein of about 1-10 mmol/l has proven to be favourable.
A further subject matter of the invention is a process for producing a renatured protein from its inactive sparingly soluble form obtainable after recombinant production in prokaryotes which is characterized in that the protein in its inactive sparingly soluble form is incubated, dissolved and derivatized with a solution of a denaturing agent in a denaturing concentration and in the presence of a disulfide component (molar ratio protein disulfide component 1:1 to 1:10000, preferably 1:1000), and the dissolved protein assumes a biologically active conformation by changing the strongly denaturing solution into a weakly or non-denaturing solution in which the disulfide bonds with the disulfide component are broken by addition of a redox system and in this manner are newly formed intramolecularly in the protein in such a way that the protein adopts a conformation in which it has its characteristic biological activity.
Such weak denaturing conditions can for example be achieved by dilution or dialysis preferably in the presence of a reducing agent. Weakly denaturing conditions are, in contrast to strongly denaturing conditions, those conditions under which the protein is able to adopt its active conformation and be stable in this conformation. Under strongly denaturing conditions the protein is unstable in this form and tends to denature i.e. to lose its stable three-dimensional structure and the energetically favourable disulfide bond. Strongly denaturing conditions exist for example in solutions of 4-9 mol/l guanidine hydrochloride. Weakly denaturing conditions exist for example between 0.1 and 2 mol/l guanidine hydrochloride. It is also expedient to add arginine at a concentration between 0.1 and 1 mol/l during the renaturation.
The activity of the protein is understood as a biological activity of the protein. If it is a naturally occurring protein or a derivative of a natural protein, its biological activity can be determined by means of the immunological, cell-biological or catalytic properties of the protein.
The activation (renaturation) is preferably carried out at a GSH concentration of 0.1-20 mmol/l, a GSSG concentration of 0.01-10 mmol/l without a denaturing agent or at a non-denaturing concentration of a denaturing agent and the reactivation is preferably carried out over a period of 1-300 hours. In this case the GSH concentration is preferably 0.5-10 mmol/l and/or the GSSG concentration is preferably 0.1-10 mmol/l.
The process according to the invention is suitable for renaturing numerous denatured proteins and in particular recombinantly produced denatured proteins. Such proteins are for example proteases, growth factors, protein hormones, cytokines, plasminogen activators, factor Xa and in particular neurotrophins. Neurotrophins are proteins which are found especially in nerve cells and support the differentiation and the survival of nerve cells. Neurotrophins (e.g. NGF, brain derived nerve growth factor (BDNF), neurotrophins 3, 4/5, 6) are therefore valuable cell therapeutic agents for the treatment of neurodegenerative diseases such as polyneuropathies, Alzheimer""s disease or injuries to the brain and spinal cord. Human nerve growth factor (NGF) is a protein which is composed of two subunits (homodimers). The xcex2 unit has been found to have the ability to affect the growth of sensory neurones and sympathetic neurones. Mature NGF is composed of 118 amino acids, contains three disulfide bridges and is not glycosylated. Biologically active NGF is present as a dimer. The DNA and amino acid sequence of NGF is described in EP-B 0 121 338 (U.S. Pat. No. 5,169,762). However, it was not possible to obtain active protein by this process. The production of active recombinant NGF is for example described in EP-A 0 329 175, EP-A 0 370 171, in Biochem. Biophys. Res. Commun. 171 (1990) 116-122, EP-A 0 414 151, Gene 70 (1988) 57-65, EP-A 0 450 386 and Gene 85 (1989), 109-114.
Brain derived neurotrophic factor (BDNF) was described by Leibrock et al., Nature 341 (1989) 149-152. BDNF supports the survival of sensory neurones in the central nervous system and appears to be successful in the treatment of Parkinson""s disease. Recombinant BDNF can for example be produced according to WO 91/03568 in CHO cells and according to WO 92/22665 in prokaryotes.